This page explains how to select reference ligands for bias experiments while complying with the published ‘Community guidelines for GPCR ligand bias’, which also provides further information about the optimal reference ligands for each type of bias.

Pathway-bias reference ligand selection

Pathway-bias studies uses a reference ligand for bias which is has balanced signaling across the compared pathways in the given biological and experimental system used. These ligands can be found in the Biased Signaling Atlas’ browser for Pathway-preferring ligands by applying a cut-off to the “Relative pathway activity (log(Emax/EC50))” which should be close to 0 (means pathway-balanced instead of pathway-preferring signaling). To increase the chance that at least one of the viewed ligands are pathway-balanced also in your experiments, it is suggested to pick and test several ligands and to prioritize ligands have been tested in similar biological and experimental systems.




Upon own testing of alternative potential reference ligand for pathway-bias, making a bias plot is a good tool to identify the most pathway-balanced ligand to select as reference.

Physiology-bias reference ligand selection

Physiology-bias studies uses a reference ligand for bias which is an endogenous ligand. These ligands can be found in GPCRdb’s browser for Endogenous ligands. If your receptor of interest has multiple endogenous ligands, community guidelines recommends using the “principal” endogenous ligand and not “secondary” endogenous ligands, which can be filtered out in this browser. This is because secondary endogenous ligands can also be physiology-biased relative to the principal endogenous ligand.

Separate reference ligand for efficacy

If the reference ligand for bias is not a full agonist (does not reach the maximum efficacy of the assay), a separate reference ligand should be used for efficacy measures. If needed, each pathway can use a distinct reference ligand for efficacy as long as the efficacies of all ligands tested for bias, and the reference ligand for bias, are measured relative the same reference ligand for efficacy. For further information, see section “1. The choice of reference ligand …” in the community guidelines.




Example of a reference ligand for bias which is a partial agonist (does not reach maximum efficacy) and therefore needs to be complemented with a separate reference ligand for efficacy.